Small interfering RNAs (siRNA) are potent reagents for directed post-transcriptional gene silencing (Hannon, G. J., 2002). siRNAs are double-stranded molecules typically 21 to 25 nucleotides (nt) in length, which trigger RNA interference (RNAi), resulting in post-transcriptional message degradation (Elbashir, S. M., et al., 2001) and inhibition of viral propagation (Andino, R., 2003). RNAi has emerged as an immensely important and popular method to elicit post-transcriptional, sequence-specific silencing of gene expression and is a major new genetic tool for investigating mammalian cells. RNAi is initiated by exposing cells to dsRNA either via transfection or endogenous expression. Long double-stranded (ds) RNAs are processed into siRNAs by dicer, a ribonuclease of the Rnase III family. These siRNAs form a complex known as the RNA Induced Silencing Complex or RISC, which functions in homologous target RNA destruction (Montgomery, M. K., 2004).
The use of exogenously supplied siRNAs for targeted RNA knockdowns has become widespread (Elbashir, S. M., et al., 2001). The exogenous RNAs can be manufactured synthetically. However, when synthetic siRNAs are used for gene silencing, the costs can be substantial because of variations in siRNA efficacies. An alternative to chemically synthesized siRNAs are siRNAs produced by bacteriophage T7 RNA polymerase. These siRNAs are made by in vitro transcription mediated by bacteriophage promoters from linearized DNA templates. In vitro transcription using bacteriophage T7 RNA polymerase has been shown to produce highly active siRNAs (Sohail, M., et al., 2003; Donze, O. and Picard, D., 2002).
The interferon (IFN) system is one of the body's first lines of defense against viruses (Samuel, C. E., 2004). IFN was discovered as an antiviral agent by Isaacs and Lindenmann during studies on virus interference, where they observed that cells infected with influenza virus secrete a factor that mediates the transfer of a virus-resistant state active against both the inducing virus and other viruses as well (Samuel, C. E., 2004). Double-stranded RNA (dsRNA) is known to play an important role in the IFN system (Samuel, C. E., 2001). It is known that synthetic dsRNAs and RNAs with double-stranded character produced during viral infections have the capacity to be potent inducers of IFN (Stewart, W. E., 1979; Marcus, P. I., 1983).
The early recognition of invasive pathogens by innate sensing is the most important defense mechanism of the immune system (Beutler, B., 2004a; Boehme, K. W. and Compton, T., 2004). Viral infection of mammalian cells results in activation of an innate immune response which is mediated by interferons and cytokines that concomitantly inhibit viral replication (Malmgaard, L., 2004). Several Toll-Like Receptors (TLRs) have been identified in humans and mice and are known to be expressed predominantly on cell types which are first to encounter intracellular pathogens (Boehme, K. W. and Compton, T., 2004). Double stranded RNA (dsRNA), including the synthetic analog poly inosine-poly cytosine (Poly IC), is known to activate TLR3, a cellular receptor that recognizes and initiates a potent anti-viral response by producing interferons (Alexopoulou, L., et al., 2001). Similarly, single stranded RNA (ssRNA), which includes the genomes of several viral RNA species, has been shown to interact with and activate TLR7 and TLR8 (Lund, J. M., et al., 2004; Diebold, S. S., et al., 2004; Heil, F., et al., 2004; Hornung, V., et al., 2005). dsRNAs can be easily distinguished intracellularly as viral replication intermediates, however, it remains elusive how a simple ssRNA motif recognized by TLR7 and 8 is discerned by the cell to be either viral (exogenous) or endogenous in origin (Boehme, K. W. and Compton, T., 2004). Considering that TLRs are cell type specific and are present within unique localized intracellular compartments, recognition of dsRNA and/or ssRNA offers an important innate defense mechanism against viral infection along with the recognition of CpG DNA motifs and/or envelope glycoproteins (Boehme, K. W. and Compton, T., 2004; Beutler, B., 2004b)
RNAi-mediated gene silencing in mammalian cells requires siRNAs of sufficiently small size to circumvent potential sequence-independent, nonspecific changes in gene expression attributable to the induction or action of interferons. Sledz, C. A., et al. (2003) found that transfection of siRNAs results in interferon (IFN)-mediated activation of the Jak-Stat pathway and global upregulation of IFN-stimulated genes. The authors showed that by using cell lines deficient in specific components mediating IFN action that the RNAi mechanism itself is independent of the interferon system. The authors characterized their finding as showing the “broad and complicating effects” of siRNAs beyond the selective silencing of target genes when introduced into cells. Similarly, Bridge, A. J., et al. (2003) reported that although siRNAs were thought to be too short to induce interferon expression, a commonly used shRNA construct was found to induce an interferon response. The authors advise as a “simple precaution to limit the risk of inducing an interferon response” to use the lowest effective dose of shRNA vector.
Although the anti-viral activities of interferons are well studied (Samuel, C. E., 2001), nobody has recognized in connection with RNAi the uses and advantages, as opposed to the risks, of interferon induction by RNAi molecules, independent of the RNAi effect, to provide anti-viral and other effects, such as anti-cancer effects. Moreover, until now, nobody is believed to have discovered the role of the triphosphate, in particular the 5-triphosphate produced on RNAi molecules in vitro, for inducing interferon and eliciting anti-viral and other medically useful responses.